1. Volume 109, Issue 4, Pages 497-508 (May 2002)
The VPAC2 Receptor Is Essential for Circadian Function in the Mouse Suprachiasmatic Nuclei 
Anthony J. Harmar, Hugh M. Marston, Sanbing Shen, Christopher Spratt, Katrine M. West, W.John Sheward, Christine F. Morrison, Julia R. Dorin, Hugh D. Piggins, Jean-Claude Reubi, John S. Kelly, Elizabeth S. Maywood, Michael H. Hastings 
Cell 
Volume 109, Issue 4, Pages (May 2002)
DOI: /S (02)

2. Figure 1
Generation of Vipr2−/− Mice and Binding of Ro in the SCN
Schematic diagram of (A) wild-type allele, (B) targeting vector, and (C) targeted allele showing restriction sites for EcoRI (E), EheI (Eh) ScaI (Sc), SacII (Sa), and XbaI (X) and positions of probes A and B and primers P1, P2, P3, and P4. EcoRI digests of genomic DNA from ES cells hybridized with probe A (D) and probe B (E).
Amplification of mouse genomic DNA by primer pair P1 and P2 (F) and P3 and P4 (G).
Coronal sections of the brain from wt (H, I, and J) and Vipr2-/- (K, L, and M) mice were incubated with labeled 125I-Ro (H) and (K) are autoradiograms showing total binding of 125I-Ro , while (I) and (L) show nonspecific binding (in the presence of 20 nM unlabeled Ro ). (J) and (M) are the hematoxylin-eosin-stained sections equivalent to (H) and (K), respectively. Strong labeling of SCN is seen in wt mice only (H).
Cell  , DOI: ( /S (02) )

3. Figure 2 Wheel-Running Activity in Wild-Type and Vipr2−/− Mice
Representative profiles of locomotor activity in Vipr2−/− (A and C) and wt (B and D) mice are presented in double-plotted format. Periods of darkness are shaded. For the first 14 days, animals were exposed to a 12:12 hr, L:DR cycle (darkness from 19:00 to 07:00). On day 14, the cycle was advanced by 8 hr (darkness from 11:00 to 23:00) via a shortened light period of 4 hr. On day 26, the cycle was returned to the original lighting regime (darkness 19:00 to 07:00) via a light period of 20 hr. From day 40, animals were maintained in constant darkness. Following 33 days of DR:DR, the original cycle was reimposed and animals were twice transferred into DR:DR to explore the effects of brief periods of light during circadian day (days 51–104; data not shown). For the last 17 days (days 110–126), an additional five Vipr2−/− mice (KO11–KO15) and one additional wt animal (WT15) were added to the study. Under a 19:00 to 07:00 L:DR cycle, three 2-hr periods of dim red light were interpolated into subjective day at 4 day intervals, the first at 13:00, the second at 11:00, and the third at 16:00. Mean activity profiles from 13 wt (E and G) and 10 Vipr2−/− (F and H) mice under either L:DR (E and F) or DR:DR (G and H) conditions are plotted in black with representative plots from individual animals in gray. The mutant mice exhibited less running wheel activity than the wt under both L:DR and DR:DR. Activity in both wt and Vipr2−/− mice correlated well with the L:DR cycle, but under DR:DR the peak of activity in Vipr2−/− mice was in antiphase to the original L:DR cycle. The mean activity counts per 6 min time bin are plotted against circadian time or zeitgeber time (lights on at ZT0). In (E) and (F), the bar at the top indicates the dark period in black and the light period in white; in (G) and (H), the bar at the top indicates subjective night in black and subjective day in gray.
Cell  , DOI: ( /S (02) )

4. Figure 3
Expression of Clock Genes under a Light:Dark Schedule Is Attenuated in Vipr2−/− Mice
Coronal brain sections from wt (filled circles) and Vipr2−/− (open circles) mice sacrificed every 6 hr under a 12 hr light:12 hr darkness lighting regime were hybridized with riboprobes encoding mPer2 (A, E, and F), mPer1 (B), mBmal1 (C), and mCry1 (D), and hybridization in the SCN (A–D), motor cortex (E), and striatum (F) was quantified as described in Experimental Procedures. The bars at the top indicate the dark period in black and the light period in white. In the SCN (A–D), two-way ANOVA revealed significant interactions between genotype and time of sampling for all four genes: mPer1 F3,16 = 9.07, p = 0.001, mPer2 F3,16 = 36.07, p < , mBmal1 F3,16 = 10.79, p = , and mCry1 F3,16 = 11.63, p = Post hoc analysis confirmed that, although expression of mPer2, mPer1, mBmal1, and mCry1 varied with time in both wt and Vipr2−/− mice, the rhythms of expression of all four genes were significantly attenuated in amplitude in the mutant animals. In motor cortex (E) and striatum (F), two-way ANOVA of the LD data revealed a significant main effect of time but not of genotype (F[3,16] = 15.78, p < in motor cortex; F3,16 = 15.63, P < in striatum). Post hoc analysis confirmed that expression was significantly rhythmic in both wt and Vipr2−/− mice.
Cell  , DOI: ( /S (02) )

5. Figure 4
Circadian Rhythmicity in Expression of Clock Genes and AVP in Wild-Type and Vipr2−/− Mice
Coronal brain sections from wt (filled circles) and Vipr2−/− (open circles) mice sacrificed every 5 hr in constant dim red light were hybridized with riboprobes encoding mPer2 (A, F, and G), mPer1 (B), mBmal1 (C), mCry1 (D), and AVP (E). Hybridization in the SCN (A–E), motor cortex (F), and striatum (G) was quantified as described in Experimental Procedures. The bars at the top indicate subjective night in black and subjective day in gray. In the SCN, two-way ANOVA revealed significant interactions between genotype and time of sampling for all five genes: mPer1 F4,20 = 35.92, p < , mPer2 F4,20 = 15.26, p < , mBmal1 F4,20 = 5.89, p = 0.003, mCry1 F4,20 = 16.26, p < , AVP F4,20 = 12.2, p < Post hoc analysis confirmed that, although expression of all five genes varied with time in wt mice, only mBmal1 was significantly rhythmic in Vipr2−/− mice F4,10 = 3.6, p = ANOVA revealed significant rhythmicity in mPer2 expression in both the striatum F5,9 = 12.93, p < 0.01 and motor cortex F5,9 = 9.93, p < 0.01 of wt mice but not in the Vipr2−/− mice.
Cell  , DOI: ( /S (02) )

6. Figure 5
Circadian Rhythmicity in Expression of Clock Genes and of AVP Is Lost in Vipr2−/− Mice
Brains from wt and Vipr2−/− mice were collected across the second cycle in DR:DR and processed by in situ hybridization (A and B) or by immunocytochemistry (C and D) to monitor circadian patterns of clock gene expression.
(A) Dark-field photomicrographs of emulsion-dipped autoradiographs showing high levels of expression of mPer2 mRNA in the SCN at CT 10 and low expression at CT20 in wt (+/+) mice. This circadian rhythmicity is lost in Vipr2−/− (−/−) mice with levels of expression of mPer2 mRNA being low at both CT 10 and CT 20.
(B) Photomicrographs of film autoradiographs showing a high level of expression of AVP mRNA in the SCN at CT 10 and low expression at CT 20. This circadian rhythmicity is lost in Vipr2−/− mice with levels of expression of AVP mRNA being low at both time points. Levels of expression of AVP mRNA in the supraoptic (SON) and paraventricular (PVN) nuclei were not affected by circadian time or the mutation.
(C) mPER1 immunoreactivity in representative coronal sections of the SCN of wt and Vipr2−/− mice at CT 12 and CT 24.
(D) mPER2 immunoreactivity in representative coronal sections of the SCN of wt and Vipr2−/− mice at CT 12 and CT 24. The scale bars for (A), (C), and (D) equal 100 μm; the scale bar for (B) equals 500 μm.
Cell  , DOI: ( /S (02) )

7. Figure 6
Induction of mPER1 and mPER2 Immunoreactivity by Light in the SCN of Wild-Type But Not Vipr2−/− Mice
Mutant and wt mice exhibiting stable activity/rest cycles under a 12 hr:12 hr L:DR cycle were either exposed to light for 6 hr, starting 2 hr after lights off (ZT14), or maintained in darkness. The number of nuclei in the SCN immunostaining for mPER1 and mPER2 immunoreactivity was counted. Two-way ANOVA revealed significant interactions between genotype and treatment for both mPER1 F1,12 = , p < and mPER2 F1,12 = 10.82, p = Post hoc analysis revealed that the number of nuclei immunostaining for both mPER1 and mPER2 was significantly elevated following 6 hr of light only in the wt animals.
Cell  , DOI: ( /S (02) )